Understanding the mechanisms of cell migration is a fundamental question in cell, developmental and cancer biology. Cell motility is influenced by a complex interplay among intracellular mechanics and signaling, and the physical cues of the microenvironment. Our knowledge on the mechanisms of cell migration stems primarily from in vitro studies using two- dimensional (2D) surfaces. Cell locomotion in 2D is driven by cycles of actin protrusion, integrin-mediated adhesion and myosin-dependent contraction. However, cells in vivo migrate within 3D extracellular matrices and through 3D pre-existing longitudinal channels created by various anatomic structures. Accumulating evidence suggests that the physical confinement affects the regulation and mechanisms of cell locomotion. For instance, myosin contractility and ?1 integrin- dependent adhesion are dispensable in cell migration through confined spaces, which persists even when F-actin is disrupted. Thus, an alternative mechanism is at play for cells migrating in narrow microchannels. We recently proposed a new mechanism of cell motility in confined spaces that is described by the osmotic engine model (OEM). According to OEM, a cell migrating in a narrow channel establishes a spatial gradient of aquaporins (AQPs), ion channels and pumps in the cell membrane, so that there is a net inflow of water at the leading edge and a net outflow of water at the cell trailing edge. We hypothesize that cells can use different mechanisms (actomyosin-based and water permeation-based) depending on the physical cues of the microenvironment. We herein propose to develop an integrated experimental and theoretical approach to delineate the mechanisms of cell entry and migration in confined spaces. Experimental work will directly interact with theory and modeling throughout our proposed studies. In Aim 1, we propose to directly measure water uptake by cells migrating through confined spaces, and decipher the molecular mechanisms of water permeation and the role of mechanosensitive (MS) ion channels in confined migration. In conjunction with experimental work, we will develop a comprehensive molecular model of OEM integrating the roles of AQPs, membrane voltages and ion channels and pumps in cell migration in narrow channels. Because confined migration largely depends on microtubule (MT) dynamics, we will determine the role of MT molecular motors and their synergistic effects with actin polymerization in establishing AQP and ion channel polarization (Aim 2). In Aim 3, we will examine the process of cell entry into physically- constricted spaces using our microfluidic device, and explore the role of cytoskeletal proteins, adhesions and membrane components during this process. Taken together, we will decipher the physical and molecular basis of a fundamentally new mechanism governing cell entry and migration in confined spaces in which AQPs, ion pumps and MS channels play key roles using a multidisciplinary approach, involving state-of-the-art bioengineering, imaging, molecular biology tools along with mathematical modeling